1. Field of the Invention
The present invention relates generally to the analysis of chemical and biological materials and, more particularly, to a modular, multi-task immunoaffinity device secured to a peripheral box and connected to capillary electrophoresis for the isolation, enrichment, separation, identification and characterization of protein biomarkers and a large diversity of analytes found at a wide range of concentrations in simple and complex mixtures. Furthermore, when two or more devices are connected in tandem to perform microreactions in one and concentrations in the other, a powerful tool is created to generate peptide cleavage of proteins and further purification and concentration of one or more resulting peptides. In addition, the present invention relates to an analyte concentrator-microreactor device connected as a functional and integral component of a cartridge-cassette.
2. Description of Related Art
Technology in the twenty-first century is shrinking at a rather rapid rate. As a result, more and more advancements are taking place at the cellular, molecular and atomic level. With scientific understanding growing, it is becoming possible to engineer the smallest devices and applications to help in a variety of fields. One of the fields that is likely to benefit greatly from miniaturization is life science. Microfabrication techniques have improved rapidly over the last decade, stimulated primarily by advancements in the microprocessor industry. Small, fast and easy-to-operate devices or instruments are needed to reduce the inherent cost and inefficiencies associated with healthcare testing in clinical laboratories. Miniaturized devices are ideally suited for using small volumes of samples and reagents, performing chemical and biochemical reactions in short periods of time, under controllable microenvironments, cost effective, environmental friendly and portables. Unfortunately, as the power of fabrication allows the manufacturing of small devices with feature sizes as small as a few microns, it becomes inevitable that the requirement of sample volumes also becomes small. As a consequence, very small sample volumes compromise the issue of sensitivity making difficult the process of isolation and quantification of analytes of interest and/or their respective modified and/or altered corresponding counterpart found at low concentrations in complex matrices, in particular in biological samples.
Due to the complexity of the samples (e.g., serum, other bodily fluids, cells, tissues) biomarker discovery remains a very challenging task. The number of chemical and biochemical substances found in the human body is not precisely known. It is certainly a large number, not to mention the enormous variety of molecules in terms of type, shape, size and function. Furthermore, the range of concentration of molecules in biological systems spans many orders of magnitude. Therefore, the use of miniaturized instrumentations and devices in the discovery of protein biomarkers has encounter a number of drawbacks. Traditionally, the analytical chemist would increase the concentration of a sample or inject more sample volume onto a column to get a better signal, thereby increasing the signal strength of one or more analytes of interest and/or their respective modified and/or altered corresponding counterpart. For those substances found at low concentrations, many kinds of amplification procedures have been developed for their analysis. Some of these techniques include immunological and/or non-immunological procedures. Many are coupled to fluorescence detectors for further enhancement of detection. Immunological assays, in particular those using fluorophore-labeled antibodies or antigens, are powerful tools used for medical diagnostics and bioanalytical chemistry because of its high detection sensitivity, non-isotopic safety, multiplexing and quantitative capabilities. In the clinic, miniaturized immuno-analytical devices are designed to move diagnostic testing out of central laboratories into sites closer to the patient.
Capillary electrophoresis (CE) technology, in the conventional and microchip format, has become a powerful tool employed in many laboratories in the search for important biomarkers of diseases. In recent years, increased emphasis has been placed on predictive biomarkers to forecast the origins or future course of toxic events or diseases caused by inflammation. Many organ-specific diseases are preceded by a long preclinical phase that is left undetected by the lack of specific and sensitive assays capable of assessing the early and advanced molecular changes that may cause dysfunction of one or more organs. Detecting a panel of “inflammatory” biomarkers in biological fluids, tissues and cells can have important predictive and confirmatory value depending on the disease and test method under consideration. Furthermore, when referred to pharmaceutical safety and efficacy, the incorporation of biomarkers into the drug development process is becoming a useful tool in the understanding of how new therapies work and allow for more accurate identification of patients who will benefit most from innovative treatments.
The major deficiency of the CE technology for the isolation and quantification of biomarkers of interest is the limits of detection (LOD), which are constrained by the small dimension of the capillary and its reduced pathlength that hinders conventional optical detection methods such as ultraviolet detection. The steps of sample purification and concentration for constituents present primarily in complex samples still remains a bottleneck in the process of sample preparation.
Analyte concentrator-microreactor (ACM) devices have been developed for selective and non-selective molecular consolidations. These analyte concentrator-microreactor (ACM) devices, which are used on-line with a capillary tube or capillary channel, have been described in U.S. Pat. Nos. 5,202,010; 6,406,604; 7,329,388; 7,736,480; 7,811,436; 8,007,724; 8,007,725; and 8,030,092 which are incorporated by references in this disclosure. U.S. Pat. No. 5,741,639 discloses the use of molecular recognition elements. U.S. Pat. No. 5,800,692 discloses the use of a pre-separation membrane for concentrating a sample. U.S. Pat. No. 7,407,568 discloses the use of sol-gel coatings for on-line preconcentration in capillary electrophoresis. U.S. Pat. No. 7,828,948 discloses the use of preconcentration and separation of analytes in microchannels. U.S. Pat. No. 7,959,861 discloses the use of integrated affinity microcolumns and affinity capillary electrophoresis.
While these devices and/or methods fulfill their respective, particular objectives and requirements, it is desirable to provide a new modular and multi-task analyte concentrator-microreactor (ACM) device secured to a portable and interchangeable box that can be mounted peripherally to a capillary electrophoresis instrument for performing on-line affinity capture, purification, preconcentration, microreactions, separation, detection, quantification, identification and characterization of analytes of interest and/or their respective modified and/or altered corresponding counterpart.
Based on these deficiencies, there exists a need for a modular and multi-task device, secured to a portable and interchangeable box, coupled to capillary electrophoresis instrument for affinity purification and having increase sensitivity for polymeric macromolecules and their smaller constituents, in particular proteins and peptides present in complex biological samples.
In these respects, the Modular and Multi-Task analyte concentrator-microreactor (ACM) Device according to the present invention substantially departs from the conventional concepts and designs of the prior art, and in so doing provides a device that when secured to a box body can become portable and interchangeable and capable of being mounted peripherally to capillary electrophoresis or to another instrument used in separation sciences.
It is also desirable to provide an ACM device that can be incorporated as a functional and integral component of an existing or custom-made cartridge-cassette utilized in laboratory-made or commercial instruments. The ACM device can have miniaturized valves to control the direction of the fluid. A cleaning buffer, conditioning buffer, and a sample fluid can be introduced through a transport capillary to avoid contamination with a separation capillary. The operation can be manual or automated, independent or incorporated as an integral part of an automated circuit of the instrument.